PROJECT SUMMARY Increasing evidence indicates that three-dimensional (3D) genome organization is required to regulate gene function and its alterations are associated with many diseases. The genome is organized into compartments that align with the temporal order of DNA replication (replication timing ? RT). However, little is known about the mechanisms underlying 3D genome organization. Recently, we identified cis elements of RT and 3D genome organization control (early replicating control elements ? ERCEs) in murine embryonic stem cells. ERCEs are enriched in enhancer epigenetic marks, form strong chromatin interactions and are bound by pluripotency-specific transcription factors. Moreover, I developed an integrative model of gene regulatory networks that predicts that co-occupancy of cell type-specific transcription factors regulate RT. Here, we will study what are the regulatory elements of genome organization in human differentiated cell types, investigate how trans-acting factors control these elements, and define how 3D genome organization is remodeled during development and evolution. Our central hypothesis is that co-occupancy of cell type-specific transcription factors at ERCEs is required to regulate the 3D genome organization. To test this hypothesis, we will delete and insert candidate ERCEs into the genome of human differentiated cell types and test their effect on RT, 3D genome organization and gene expression. We will track ERCE activation using highly-synchronous human embryonic stem cells differentiation systems. Finally, we will analyze 3D genome organization evolution using primary cells derived from different species. My laboratory is uniquely positioned to perform the proposed research with broad expertise in the experimental design and analysis of 3D genome organization during human cell fate commitment. Moreover, numerous resources available at the University of Minnesota will facilitate the success of this project, including state-of-the-art technologies for genome editing (Genome Engineering Shared Resource), next-generation sequencing (Genomics Center), stem cells (Stem Cell Institute), imaging (Imaging Center) and bioinformatic tools (Minnesota Supercomputing Institute). We expect that our work will contribute significantly to understand the fundamental principles of genome organization and its relationship to gene function. The proposed research combines several innovative aspects such as integrative computational models to predict regulatory elements of large-scale chromatin organization, genome engineering technologies and optimized differentiation protocols of human embryonic stem cells to dissect the mechanisms that control 3D genome organization during development and evolution.